The relationship among DNA methylation, DNA conformation and gene activity will be examined during mammalian spermatogenesis. Transcriptionally active somatic genes typically are associated both with DNA hypomethylation and with DNA hypersensitivity to certain nucleases. However, it is not known whether these structural signals are important during spermatogenesis, a period of cellular differentiation when gene transcription eventually ceases as protamines replace histones on the DNA and the chromatin condenses. cDNA libraries to spermatogenic-specific genes will be generated in order to map any hypomethylated and hypersensitive point sites in these genes. Since these genes are expressed only during spermatogenesis, it might be expected that hypomethylated point sites will be found in spermatogenic DNA without corresponding hypersensitive sites in somatic DNA. The changes in chromatin structure can be directly correlated with gene activity by examining specific stages of spermatogenesis. By this method the state of the DNA before, during, and after transcription of each gene can be ascertained. In particular, it would be important to determine if the control of gene expression is under different signals in spermatogenic cells then is found in somatic cells. The possible effects of hypomethylation on gene expression at fertilization will be investigated, in addition. Hypomethylation of genes during spermatogenesis may be a mechanism for signaling the transcription of these genes early in embryogenesis. The cDNA probes will be used to determine if any genes remain hypomethylated in the mature sperm when transcription has ceased. If so, the transcriptional state of these genes in the embryo will be examined. Finally, genes that are hypomethylated early in spermatogenesis and are retained in that state throughout differentiation may be important in "marking" the germ cell line in the embryo. These genes can be followed into the embryo to determine if any differences are apparent in the methylation pattern when the germ line and non-germ line tissues are analyzed. The experiments in this proposal will provide the foundation for a long-term molecular examination of mammalian spermatogenesis.